Abstract

Claudin 18 (CLDN18) is a tight junction protein that is highly expressed in the lung. While mice lacking CLDN18 exhibit the expected loss of epithelial integrity in the lung, these animals also have unexpectedly large lungs. In this issue of the JCI, Zhou, Flodby, and colleagues reveal that the increased lung size of Cldn18–/– mice is the result of increased type 2 alveolar epithelial (AT2) cell proliferation. This increase in proliferation was shown to be driven by translocation of the transcriptional regulator Yes-associated protein (YAP) to the nucleus and subsequent induction of proliferative pathways. CLDN18-deficent mice also had increased frequency of lung adenocarcinomas. Together, the results of this study advance our understanding of the mechanisms that likely regulate homeostasis of the normal lung as well as promote the proliferative state of malignant cells found in lung adenocarcinomas thought to originate from AT2 cells.

Apical junctional complexes (AJCs) in polarized epithelia are composed of adherens junctions (AJs) and tight junctions (TJs) (1, 2) that mediate intercellular adhesion and paracellular permeability to ions and solutes, respectively. TJs, the most apical component of AJCs, are composed of integral membrane proteins (3, 4) linked to the actin cytoskeleton via accessory proteins (5). Claudins are a family of integral membrane proteins essential for TJ formation and integrity (3, 6–8). Unique permeability properties of various epithelia are determined by cell-specific patterns of claudin expression. Targeted deletions in mice as well as human mutations have revealed novel tissue-specific biological functions of claudins in several organs including kidney (9, 10), skin (11), ear (12), and peripheral nervous system (13). Interestingly, mice with knockout of Cldn15 demonstrated an unexpected phenotype of upper small intestine enlargement (megaintestine) and increased crypt cell proliferation without evidence of inflammation (14). Both up- and downregulation of claudin expression have been observed in a number of cancers, although how these changes contribute to carcinogenesis and/or neoplastic progression remains controversial (15–18). These studies suggest that, in addition to traditional roles in regulating epithelial barrier function and polarity, claudins also regulate cell functions such as proliferation that might contribute to tumorigenesis.

CLDN18 is one of the most highly expressed claudin family members in lung alveolar epithelium (19, 20). It is expressed at low levels in airway epithelium and is not expressed in lung endothelium (21). Cldn18 has 2 promoters, each with a unique exon 1 spliced to common exons 2 through 5. Alternative promoter usage leads to production of lung- and stomach-specific isoforms (22). Deletion of the stomach-specific Cldn18.2 isoform in mice leads to loss of TJ strands and increased paracellular H+ leakage in the stomach, resulting in atrophic gastritis and metaplasia but without evidence of tumor formation (23). Recently generated Cldn18–/– mice with deletion of both isoforms showed increased lung permeability to ions and solutes, consistent with known roles of claudins in regulation of barrier function (24, 25).

The conserved Hippo signaling pathway is a key regulator of organ size, stem/progenitor cell function, and tumorigenesis that exerts opposing effects on cell proliferation and apoptosis by controlling cellular localization of the downstream transcriptional coactivator Yes-associated protein (YAP) (26, 27). Cellular localization of YAP is determined by activity of the core Hippo kinases mammalian sterile 20–like 1/2 (MST1/2) and large tumor suppressor homolog 1/2 (LATS1/2), which phosphorylate YAP on serine residues leading to its cytoplasmic retention by 14-3-3 proteins and proteasomal degradation (26, 28). Dephosphorylated YAP translocates to the nucleus where it functions as a transcriptional coactivator of target genes (29) primarily via interactions with transcription enhancer factors 1–4 (TEF/TEAD 1–4) (30). Uncontrolled YAP activation leads to tissue overgrowth (31, 32), while increased YAP activity has been demonstrated in several cancers including lung adenocarcinoma (LuAd) (33, 34). YAP localization and activity are also regulated through interactions with membrane-associated proteins important for maintenance of cell polarity (e.g., Crb3 [ref. 35] and angiomotins [AMOTs] [refs. 36, 37]) and cell-cell contact (e.g., E-cadherin and α-catenin [refs. 38–40]) in both a Hippo-dependent and -independent manner.

Stem/progenitor cell populations in adult lung have been identified in region-specific niches along a proximal-distal axis, functioning as facultative progenitors that become activated for respiratory epithelial repair following injury (41–43). AT2 cells have been identified as progenitors of distal lung epithelium (44, 45) that are relatively quiescent under physiological conditions, becoming activated following injury. Molecular mechanisms that maintain homeostasis or activate endogenous lung stem/progenitor cells to promote adult lung repair are not yet well defined. In particular, a role for sites of cell-cell contact in modulating signals to regulate lung progenitor cell function has not been previously explored. We report in this study that, in addition to previously reported increases in lung epithelial permeability to ions and solutes, Cldn18–/– mice show enlargement of lung, stomach, and kidney, sites of CLDN18 expression. Lung parenchyma in Cldn18–/– mice is expanded with increased abundance and proliferation of AT2 cells together with activation of YAP signaling. Inhibition of YAP and overexpression of CLDN18 inhibit AT2 cell proliferation and progenitor capacity. Interestingly, aged Cldn18–/– mice showed increased propensity to develop LuAd, with stage-specific downregulation of CLDN18 in human LuAd. These results identify CLDN18 as a potentially novel regulator of YAP activity that acts to restrict progenitor cell proliferation and suggest a tumor suppressor role for CLDN18. Furthermore, since TJs become established during normal tissue morphogenesis and are frequently disrupted following injury, they suggest a mechanism whereby extracellular signals are transduced from sites of cell-cell contact to control organ size during development and regulate stem/progenitor cell proliferation and expansion following injury.

Loss of CLDN18 leads to adenocarcinoma development in aged Cldn18–/– mice and CLDN18 is downregulated in human LuAd. Alterations of CLDN18 in LuAd (49, 50) and the known role of AT2 cells as the cell of origin of LuAd led us to evaluate lung tumor development in Cldn18–/– mice. Lungs of 9- to 10-month-old Cldn18–/– mice do not show obvious tumors. However, by 18–20 months of age, approximately 80% of Cldn18–/– mice compared with approximately 12% of WT mice develop tumors (Figure 6, A and B), generally in a subpleural location and typically adenocarcinomas with papillary features and fibrovascular cores (Figure 6B). Tumor cells show increased nuclear/cytoplasmic ratio and mitotic figures (Supplemental Figure 15A). Alveolar mononuclear cells (likely macrophages) are associated with nearly all tumors (Supplemental Figure 15B). Representative micro-CT images show that some Cldn18–/– mice have more than one lung tumor at 16 months of age (Figure 6C and Supplemental Table 2) without visible tumors in WT mice. Tumor cells express AT2 cell marker SFTPC, but not club cell marker SCGB1A1 (Figure 6D), suggesting an AT2 cell origin.

Located at sites of cell-cell contact, AJC proteins are well suited to transmit extracellular signals to regulate normal tissue maintenance and restore homeostasis following disruption of the epithelial barrier (53). We previously showed a critical nonredundant role for CLDN18 in regulating alveolar epithelial TJ composition and permeability properties in Cldn18–/– mice (24). Unexpectedly, we found an increase in size of lung, stomach, and kidney, all major sites of CLDN18 expression. We focused on the lung to further explore mechanisms underlying increased organ size. We demonstrate that increased lung size is mainly the result of parenchymal expansion accompanied by increased abundance and proliferation of AT2 cells, progenitors of distal lung epithelium. Mice with deletion of Cldn18 only in AT2 cells similarly showed increases in AT2 cell number and the proportion of AT2 cells in S and G2/M phase, indicating epithelial cell specificity. These findings suggest a potentially novel role for alveolar epithelial CLDN18 in restricting stem/progenitor cell proliferation (and thereby organ size). Consistent with in vivo results, Cldn18–/– AT2 cells in 3D culture showed increased CFE, which was maintained after passaging. Lungs of Cldn18–/– mice showed increased nuclear YAP with upregulation of several YAP target genes. Cldn18–/– AT2 cells in 3D culture also showed increased nuclear YAP, while inhibition of YAP signaling decreased Cldn18–/– AT2 cell proliferation in vivo and proliferation and CFE in vitro. Conversely, overexpression of CLDN18 decreased YAP nuclear localization and CFE of Cldn18–/– AT2 cells and decreased YAP transcriptional activity. Endogenous CLDN18 interacted with both ZO-1 and p-YAP, suggesting complex formation among these proteins localized to TJs, while overexpressed CLDN18 was similarly associated with both ZO-1 and p-YAP. Colocalization of YAP and CLDN18 at sites of cell-cell contact further suggests that CLDN18 plays a role in sequestering p-YAP at TJs. Cldn18–/– mice show increased propensity to develop LuAd with age, while human LuAd show stage-dependent reduction of CLDN18 and increased nuclear YAP. These findings identify a potentially novel role for integral TJ protein CLDN18 in regulating organ size and restricting distal lung epithelial progenitor capacity and proliferation, and indicate a role for YAP activation in regulating distal lung epithelial progenitor function.

A possible role for claudins in regulation of organ size and/or cell proliferation was suggested by the phenotype of Cldn15–/– mice that showed upper small intestine enlargement with expansion of proliferating crypt cells and an increased number of crypts per villus (14), although underlying mechanisms were not explored. In the current study, we demonstrate an increase in lung size with expansion and increased proliferation of AT2 cells, uncovering a role for CLDN18 in regulation of organ size and progenitor function. In the stomach, which was also enlarged, we observed expansion of the gastric mucosa (Supplemental Figure 4) with increased proliferation (data not shown). These results differ from previous studies in Cldn18.2–/– mice in which deletion of the stomach-specific isoform led to paracellular H+ leakage, upregulation of interleukin-1β, and atrophic gastritis (23) without organ enlargement or tumorigenesis, perhaps as a result of compensatory increases in Cldn18.1. The paradigm of CLDN18 restricting progenitor cell proliferation can therefore be extended to other sites of CLDN18 expression.

AT2 cells serve as progenitors of adult distal lung, undergoing both self-renewal and transdifferentiation to give rise to AT1 cells during normal turnover and following injury (44, 45, 58). Knowledge of signaling pathways that regulate stem cell proliferation and/or differentiation during alveolar repair and regeneration is limited. A recent study implicated YAP activation in response to mechanical tension in distal lung progenitor proliferation and expansion after pneumonectomy, suggesting a role for YAP signaling in alveolar regeneration in this context (59). In the current study, we show that loss of CLDN18 leads to YAP activation and progenitor expansion in distal lung in the absence of injury, uncovering a role for YAP signaling in regulating distal lung epithelial progenitor maintenance and proliferation downstream of CLDN18 and suggesting that CLDN18 regulates AT2 cell quiescence by restricting YAP activity. Cldn18–/– AT2 cells were still able to give rise to AT1 cells in vivo and in both 2D and 3D culture in vitro, indicating that YAP activation as a result of loss of CLDN18 did not significantly affect distal lung epithelial cell fate (55). Outcome of YAP activation may thus be context dependent and distinct in conducting airways and distal lung.

The Hippo pathway is the major regulator of YAP activity (60, 61). Upstream signals that activate Hippo (and inactivate YAP) include extracellular soluble factors via G protein–coupled receptors, mechanotransduction, cell-cell contact, and polarity (28, 62), including apical polarity complexes such as Crb3 (63). There is also accumulating evidence for Hippo kinase–independent modulation of YAP activity through direct interactions with cell membrane- and cytoskeleton-associated proteins that regulate apical-basolateral polarity and cell-cell contact (26). In this regard, AJ proteins E-cadherin and α-catenin inhibit YAP signaling by both Hippo-independent and -dependent mechanisms (39, 40). Similarly, AMOTs, TJ-associated scaffolding proteins that maintain TJ integrity and epithelial polarity, regulate YAP activity leading to sequestration in cytoplasm and at TJs (64). AMOTs also promote LATS activation by serving as a scaffold that brings Hippo kinases and YAP together at TJs (65). We have demonstrated in the current study a role for integral TJ protein CLDN18 in regulating YAP activity. Co-IP demonstrates interaction among p-YAP, CLDN18, ZO-1, and p-LATS1/2 in WT AT2 cells. Furthermore, when overexpressed, CLDN18 and YAP colocalize at sites of cell-cell contact, suggesting sequestration of p-YAP at TJs. Decreased p-YAP in Cldn18–/– lungs and AT2 cells and decreased p-LATS/p-YAP interaction in membranes of Cldn18–/– AT2 cells suggest that CLDN18 is required for Hippo signaling, and furthermore that CLDN18 serves a scaffolding function that promotes interaction between the Hippo kinases and p-YAP at TJs. Loss of CLDN18 likely disrupts a membrane protein complex that includes YAP and Hippo kinases leading to increased nuclear translocation of dephosphorylated YAP. Elucidation of specific protein-protein interactions that mediate CLDN18 interaction with p-YAP/YAP will require further investigation.

Claudin expression is frequently altered in cancer (18, 66, 67), with variable up- and downregulation depending on tissue type and disease stage (15, 18, 68–70). There is thus no unifying concept as to how claudin dysregulation contributes mechanistically to carcinogenesis. CLDN18 expression is altered in both gastric cancer and LuAd, although most studies have not distinguished between expression of Cldn18.1 versus Cldn18.2 isoforms. CLDN18 was significantly downregulated in gastric cancer compared with surrounding normal gastric mucosa and its expression correlated inversely with proliferative potential at the invasive front and invasive properties of a gastric cancer cell line (71), while downregulation of CLDN18 correlated with poor survival (72). In contrast, there are reports of sustained membranous CLDN18.2 expression in gastric cancer, as well as ectopic upregulation in tumors of lung, ovary, and esophagus (73). Based on these studies, Ab-mediated therapeutic targeting of CLDN18.2 in cancers with upregulation of CLDN18.2 has been suggested (74). However, CLDN18 expression was detected in only approximately 20% of LuAd (50), with overall expression being lower in tumors than in normal lung tissue, while CLDN18.2 was expressed in only 3.7% of LuAd (74). Thus, targeting of CLDN18.2 would not be helpful for the majority of LuAd. Our analysis (Figure 6E) indicates that the CLDN18.1 isoform is downregulated in LuAd. Consistent with these findings, overexpression of CLDN18.1 was found to suppress abnormal proliferation and motility of A549 cells, a human LuAd cell line, via inhibition of PI3K/PDK1/Akt signaling (75). Therapeutic targeting of CLDN18 should therefore take into account the specific isoform that is altered in a particular subtype of LuAd.

We show that loss of CLDN18 activates YAP signaling and promotes proliferation of AT2 cells, recognized cells of origin for LuAd. Furthermore, loss of CLDN18 leads to development of LuAd in aged mice, while TCGA data show stage-specific downregulation of CLDN18.1 in LuAd. Mechanisms underlying CLDN18.1 downregulation in LuAd remain to be determined. YAP expression correlates with poor prognosis in non–small cell lung cancer (49) and YAP activity drives tumor progression in mouse models of LuAd (34). Although YAP hyperactivation regulates cellular properties important for cancer development (e.g., promotion of stemness, proliferation and dedifferentiation, and resistance to apoptosis), precise mechanisms leading to tumorigenesis as a result of YAP activation remain unclear (33). The number of stem cell divisions within a tissue correlates strongly with risk of carcinogenesis, suggesting accumulation of random mutations and genomic instability due to uncontrolled AT2 cell proliferation in Cldn18–/– mice as a potential mechanism underlying tumorigenesis (76). Our results establish an important regulatory role for TJs in general and CLDN18 in particular in restricting YAP activity to prevent uncontrolled progenitor cell proliferation and resulting tumorigenesis, while also suggesting that transient modulation of CLDN18 and/or YAP function may be of benefit by harnessing progenitor cells to promote regeneration following lung injury.

Constitutive and epithelial cell–specific Cldn18-knockout mice. Generation of Cldn18–/– mice has recently been reported (24). Sftpc+/creERT2 (Harold A. Chapman, UCSF) mice (77) crossed to Cldn18fl/fl mice were further crossed with ROSATm/Tm reporter mice (78), yielding mice with the genotype Sftpc+/creERT2;Cldn18fl/fl;ROSA+/Tm. Cldn18 knockout was induced by administration of Tmx at a dose of 100 mg/kg i.p. for 2 consecutive days. Control mice with the genotype Sftpc+/creERT2;ROSA+/Tm received the same dose of Tmx to label AT2 cells.

Tissue preparation and morphological analysis. Tissues harvested from WT and Cldn18–/– mice were prepared as previously described (24). To quantify tumors, H&E–stained lung sections from WT and Cldn18–/– mice were examined by light microscopy in a blinded fashion.

Lung volume measurement. Lung volume was measured as previously described (79). Briefly, mice were mechanically ventilated for 10 minutes with 100% oxygen. The tracheal tube was then clamped for 10 minutes to allow circulating blood to absorb oxygen and collapse airspaces. Total volume at pressures of 0, 5, 10, 15, 20, 25, and 30 cmH2O was measured using a suspension technique after inflation with PBS.

Micro-CT imaging. Lungs were fixed and inflated with 4% paraformaldehyde (PFA) at 20 cmH2O pressure overnight and then incubated through a serial ethanol gradient (50%, 70%, 80%, 90%, and 100%) followed by incubation with 100% hexamethyldisilazane overnight before air drying (80). Lung specimens were scanned at an isotropic resolution of 10 microns at 45 kVp, 200 mAs (81). CT image raw data were analyzed using AMIRA software (FEI) to create volume renderings (82). 3D segmentation to compartmentalize the lung into tissue and conducting airway was performed based on threshold of gray value difference between tissue and air. Small sections (~1 mm3) were cut from the distal portion of the lung and scanned at an isotropic resolution of 0.7 microns. Volumes of whole lung (VTlung) and conducting airway (VCairway) were measured based on CT scan contrast data of whole lung. Alveolar airspace and parenchymal fractions (Falv and Fpar) of 1-mm3 lung sections were calculated based on CT scan contrast data of the small section, assuming that the 2 fractions could be applied to the remainder of distal lung. Alveolar airspace (Valv) and parenchymal (Vpar) volume of the whole lung were calculated by multiplying (VTlung – VCairway) by Falv and Fpar, respectively. The relationship among different compartments shown in Supplemental Table 1 is presented as the following equation: VTlung = VCairway + Valv + Vpar. For radiographic measurement of lung tumors, regions of high density on acquired CT images were automatically detected, counted, and volumetrically quantified. A CT threshold (320 Hounsfield units [HU]) was used to segment lung tumor from normal lung areas.

Isolation of mouse AT2 cells. For cell isolation for 3D culture, mouse lungs were digested with elastase (4 U/ml, LS002280, Worthington Biochemical). Cells were resuspended at 106 cells in 100 μl of HEPES-buffered salt solution (HBSS) in a mixture of Abs, including anti-CD45, anti-CD31, anti-CD34, anti–SCA-1, anti-EPCAM, anti-CD24, and relevant isotype controls. AT2 cells (CD45–CD31–CD34–SCA-1–CD24–) were sorted from the EPCAM+ lung epithelial population using a MoFlo XDP (Beckman Coulter) or FACSAria (BD Biosciences) sorter as previously described (83). Data were analyzed using FlowJo software (TreeStar). For passaging, colonies were dissociated using dispase (354235, BD Biosciences) followed by magnetic selection for EPCAM+ cells. For 2D culture and cell cycle analysis, AT2 cells were isolated from WT and Cldn18–/– mice as previously described (84). For cell cycle analysis of Tm+ AT2 cells, single-cell suspensions were generated by dissociation with dispase of lungs from Sftpc+/creERT2;Cldn18fl/fl;ROSA+/Tm and control Sftpc+/creERT2;ROSA+/Tm mice.

Quantification of NKX2-1+ cells. Lung sections from WT and Cldn18–/– mice were stained for NKX2-1 with DAPI as nuclear counterstain. Five random pictures were taken using the ×40 lens of a Nikon Eclipse 80i microscope. Numbers of NKX2-1+ and total cells (DAPI+) were counted in each of 5 lung fields for each genotype. Approximately 4,000 cells from each group were counted.

Quantification of SFTPC+ and Tm+ cells. Lung sections stained with SFTPC with DAPI as nuclear counterstain were scanned with an Axio Scan.Z1 (Carl Zeiss Microscopy). The number of SFTPC+ and DAPI+ cells were counted in 2 randomly chosen regions for each genotype using Imaris software (Bitplane). Approximately 50,000 DAPI+ cells from each group were counted. For Tm+ cell counting, frozen lung sections were stained with DAPI, scanned, and counted.

EdU labeling. 5-Ethynyl-2′-deoxyuridine (EdU) was injected i.p. at 50 mg/kg body weight 24 hours prior to harvest. EdU incorporation was detected with a Click-iT Plus EdU Imaging Kit (10337, Life Technologies) following immunostaining for NKX2-1. E18 lung sections were scanned (Axio Scan.Z1) and a total of approximately 13,000 and approximately 17,000 NKX2-1+ cells were counted from each genotype using Imaris software. To determine the percentage of EdU+NKX2-1+ cells postnatally, a total of approximately 1,000 NKX2-1+ cells from 5 random fields were counted. To label proliferating MLE-15 cells, EdU (10 μM) was added to culture medium 48 hours after transfection. To label proliferating AT2 cells in 3D culture, EdU (10 μM) was added to medium 3 hours before harvesting cells.

Western analysis. Preparation of protein lysates from AT2 cells or whole lung and subsequent Western analysis were performed as previously described (86). Antigen-Ab complexes were visualized and quantified by enhanced chemiluminescence (Pierce) using a Fluor-Chem Imaging System (Model 8900, Alpha Innotech) or an Azure Imager c300 (Azure Systems). See complete unedited blots in the supplemental material.

RNA isolation, RT-PCR, and qRT-PCR. RNA extraction and cDNA synthesis were performed as previously described (86). qRT-PCR was performed with SYBR-Green reagent (Applied Biosystems) in a 7900HT Fast Real-Time PCR System (Applied Biosystems). Sequences of primers used are as follows: Cldn18 forward 5′-GACCGTTCAGACCAGGTACA-3′ and reverse 5′-GCGATGCACATCATCACTC-3′; Ccnd1 forward 5′-gcgtaccctgacaccaatct-3′ and reverse 5′-cacaacttctcggcagtcaa-3′; Cdk6 forward 5′-gcctatgggaaggtgttcaa-3′ and reverse 5′-gggctctggaactttatcca-3′; Ctgf forward 5′-CCACCCCAAACCAGTCATAA-3′ and reverse 5′-TGCTGTGCAGGTGATAAAGC-3′; and Areg forward 5′-CATCGGCATCGTTATCACAG-3′ and reverse 5′-ACAGTCCCGTTTTCTTGTCG-3′.

Cell cycle analysis. Cell cycle analysis for AT2 cells was performed using PI as previously described (87). Tm+ AT2 cells were stained with Hoechst 33323 (H3570, Thermo Fisher Scientific). Flow cytometry analysis was performed using a FACSAria or LSRII flow cytometer (BD Biosciences) and ModFit LT Version 4 or 5 software.

VP treatment. VP, brand name Visudyne (1786, Selleckchem) was dissolved in dimethyl sulfoxide (DMSO) at 100 mg/ml and diluted in PBS to 15 mg/ml. VP was administered i.p. at 100 mg/kg body weight daily from P4 to P11, and lungs were harvested at P12 for evaluation of cell proliferation. To examine dry weight/body weight ratios, pregnant females were injected (100 mg/kg body weight) at E13, E15, E17, and E19 followed by postnatal injection into pups from P4 and then every other day up to P14 at the same dose of VP. Lungs were harvested on P16. VP was added to AT2 cells in 3D culture from day 2 at a concentration of 0.75 μM. 3D cultures were imaged and harvested for staining at day 14.

Transient transfections in MLE-15 cells. Twenty-four hours after seeding, 0.5 μg 5xUAS-Luc, 0.3 μg GAL4-TEAD (from Jiandie Lin, University of Michigan), and 30 ng pEGFP-C3-Yap (from Marius Sudol, National University of Singapore) or pCMV-Flag-YAP5SA were cotransfected into MLE-15 cells together with 0.25 μg pCMV6-AC-CLDN18-GFP or control pCMV6-AC-CLDN18-GFP (Origene). Luciferase assay was performed 48 hours following transfection and normalized to protein concentration.

Phos-tag Western blotting. Phos-tag gels were prepared according to the manufacturer’s instructions (300-93523, Wako Chemicals). Protein separation and immunoblotting were performed using standard protocols for Western blotting.

Statistics. Values are the mean ± SEM. Significance (P < 0.05) for 3 or more group means with 1 and 2 factors was determined by 1-way and 2-way ANOVA, respectively. Post hoc analyses were performed with Bonferroni’s corrections. Two group means and a 2 × 2 contingency table were compared for significance using 2-sided t tests and Fisher’s exact test, respectively. Z tests were used to determine if ratiometric (i.e., normalized) data were different from control. Statistical analyses were performed using SPSS version 19 (IBM) and Microsoft Office Excel 2013.